We will develop vaccines against hookworms using RNA-seq and comparative genomics to identify targets essential for infection. Hookworms such as Ancylostoma duodenale and Necator americanus infect over 400 million human beings, stunting and impoverishing them. Existing drugs are only partially effective, and no reliably effective vaccines against hookworms exist. The zoonotic hookworm Ancylostoma ceylanicum easily infects both humans and other mammals (such as golden hamsters). It is therefore a useful experimental model in which to test possible treatments for hookworm disease. Our first hypothesis is that by using transcriptional analysis of genes upregulated during infection by both A. ceylanicum and the related parasite Haemonchus contortus, we have identified a novel set of secreted proteases and protease inhibitors that are essential for parasite survival and that are promising vaccine targets. Our second hypothesis is that we can use RNA-seq and tissue-specific expression to identify a deeper set of vaccine targets, encoded by genes whose products are required for hookworms to negate the host immune system and that are accessible via vaccination. We have recently generated and analyzed an A. ceylanicum genomic sequence of 313 Mb containing ~27,000 genes, along with RNA-seq data during an A. ceylanicum infection from third-stage larvae to fully mature adults. This has already provided us with our top priority protease and protease inhibitor vaccine candidates. Our first aim will be to prioritize these candidates by tissue-specific expression, synthesize them in the yeast Pichia pastoris, and then test them in mixtures as vaccines against A. ceylanicum. Although previous efforts at vaccination with crude mixtures of nematode proteins from killed or weakened parasites have given promising results, vaccinations with single proteins from individual parasite genes have generally failed. We suspect that multiple gene groups expressed during infection by a single parasite genome can be collectively expressed and coinjected to elicit effective immunity. T cell responses to successful vaccine formulations will be characterized. Our second aim will be to identify immunoresponsive intestinal hookworm proteins as further vaccine candidates. We propose to perform additional RNA-seq on dissected intestines and non-intestinal tissues, from A. ceylanicum infecting golden hamsters with either normal or suppressed immune systems. We will use bioinformatics to look for genes encoding secreted or membrane-bound proteins, which, during infection, are either interacting with the host's immune system and/or robustly translated in the parasite's intestines. These are good candidates for proteins used by the parasite for neutralization of the immune system. We will further prioritize candidate secreted and membrane-bound gene products by their conservation in the human hookworm Necator americanus and in H. contortus. Vaccination will begin with the top one or two candidates, to be expanded upon in a subsequent R01.